Composition comprising at least one 15-pgdh inhibitor

ABSTRACT

The invention relates to a cosmetic composition containing at least one 15-hydroxy-prostaglandin dehydrogenase inhibitor and cosmetically acceptable excipients. It also relates to a method of cosmetic treatment for promoting the growth and/or preventing or delaying the loss of hair, and the use of a 15-hydroxyprostaglandin dehydrogenase inhibitor for the preparation of a composition intended for controlling hair loss and/or for promoting hair regrowth.

CROSS-REFERENCE TO EARLIER UNITED STATES APPLICATIONS

This application is a divisional application of prior copending U.S.patent application Ser. No. 10/420,831, filed Apr. 23, 2003, nowallowed, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/387,420, filed Jun. 11, 2002, and of U.S.Provisional Application No. 60/425,330, filed Nov. 12, 2002, the entirecontents of each earlier U.S. application being hereby incorporated byreference.

CROSS-REFERENCE TO FOREIGN PRIORITY APPLICATIONS

This application claims priority under 35 U.S.C. § 119 of FR 02/05067and FR 02/13461, filed in France on Apr. 23, 2002 and Oct. 28, 2002,respectively, the entire contents of each of which is herebyincorporated by reference.

The present invention relates to the use of compounds for thepreparation of compositions intended for inducing or stimulating thegrowth of keratinous fibres and in particular human hair and eyelashes.The invention relates to novel compositions for controlling hair lossand/or promoting hair regrowth. It also relates to a method of cosmetictreatment for controlling the loss of keratinous fibres, such as hair oreyelashes, and/or promoting the growth of keratinous fibres, inparticular hair growth or regrowth, in particular for maintaining and/orincreasing hair density in humans.

In human beings, the growth of hair and its renewal are mainlydetermined by the activity of the hair follicles and their cutaneousand/or connective environment. Their activity is cyclic and comprisesessentially three phases, namely the anagen phase, the catagen phase andthe telogen phase.

The active anagen phase or growth phase, which lasts for several yearsand during which the hair grows longer, is followed by a very short andtransient catagen phase which lasts for a few weeks. During this phase,the hair undergoes an involution, the follicle is atrophied and itsdermal implantation appears increasingly high.

The terminal phase, termed telogen phase, which lasts for a few months,corresponds to a resting period for the follicle and the hair finallyfalls out. After this resting phase, a new follicle is regenerated, inplace, and another cycle begins.

The hair is therefore continuously renewed, and of the approximately 150000 hair strands which make up the hair, at each instant, approximately10% of them are in the resting phase and will therefore be replaced in afew months.

The natural loss of hair can be estimated, on average, at a few hundredhair strands per day for a normal physiological state. This process ofpermanent physical renewal undergoes a natural evolution during ageing,the hair becomes thinner, and its cycles shorter.

In addition, various causes can result in a transient or permanent lossof hair.

This may involve hair loss and damage towards the end of pregnancy(postpartum effluvium), during states of undernourishment or of dietaryimbalances, or during states of asthenia or of hormonal dysfunction asmay 90 be the case during or towards the end of menopause. It may alsoinvolve hair loss or damage in relation to seasonal phenomena.

It may also involve alopecia which is essentially a disruption in hairrenewal which causes, in the first instance, the acceleration of thefrequency of the cycles at the expense of the quality of the hair andthen of its quantity. Successive growth cycles result in increasinglythin and increasingly short hair, gradually changing to a nonpigmenteddown. Some areas are preferentially affected, in particular the temporalor frontal sinuses in men, and in women a diffuse alopecia of the vertexis observed.

The term alopecia also covers a whole family of impairments of the hairfollicles of which the final consequence is the partial or generalpermanent loss of hair.

It involves more particularly androgenic alopecia. In a large number ofcases, premature hair loss occurs in genetically predisposed subjects,it then involves androchronogenetic alopecia; this form of alopeciaapplies to men in particular.

It is moreover known that certain factors such as hormonal imbalance,physiological stress and malnutrition can intensify the phenomenon.

In certain dermatoses of the scalp with an inflammatory characteristic,such as for example psoriasis or seborrhoeic dermatitis, hair loss canbe greatly intensified or can cause greatly disrupted hair cycles.

Compositions which make it possible to suppress or reduce alopecia, andin particular to induce or stimulate growth of hair or to reduce itsloss, have been sought for many years in the cosmetics or pharmaceuticalindustry.

For this purpose, a large number of compositions comprising widelyvarying active agents have already been proposed, such as for example2,4-diamino-6-piperidinopyrimidine 3-oxide or “Minoxidil” described inU.S. Pat. No. 4,139,619 and U.S. Pat. No. 4,596,812 or its manyderivatives such as those described for example in patent applicationsEP 0353123, EP 0356271, EP 0408442, EP 0522964, EP 0420707, EP 0459890and EP 0519819.

Clinical studies have demonstrated that PGF2α analogues had the propertyof causing the growth of body hair and of eyelashes in humans and inanimals (Murray A and Johnstone M D, 1997. Am J Opht, 124(4), 544-547).In humans, trials carried out on the scalp have shown that aprostaglandin E2 analogue (viprostol) had the property of increasinghair density (Roenigk H H., 1988, Clinic Dermatol, 6(4), 119-121).

Patent WO 98/33497 describes pharmaceutical compositions containingprostaglandins or derivatives of prostaglandins intended for controllinghair loss in humans. Types A2, F2α and E2 prostaglandins are preferred.

However, prostaglandins are molecules which have a very short biologicalhalf-life period and which act in an autocrine or paracrine manner,reflecting a labile character due to a very active local metabolism ofthe prostaglandins (Narumiya S et al., 1999, Physiol Rev, 79(4),1193-1226).

It therefore appears to be important, in order to maintain and/orincrease hair density in humans, to preserve the endogenous reserves ofPGF2α as well as of PGE2 of the different compartments of the hairfollicle or of its close cutaneous environment.

A solution which gives good results is the administration oflipoxygenase inhibiting and/or cyclooxygenase inducing compounds so asto promote hair growth; a hypothesis is that the administration of suchcompounds orients the metabolism of fatty acids towards the endogenoussynthesis of prostaglandins, rather than using other pathways.

However, to further improve the results, it would be desirable to beable to prolong the activity of the prostaglandins involved in thegrowth and the maintenance of the activity of the hair follicle.

It is moreover well known that the programmes of differentiation of thekeratinocytes of the epidermis and of the hair follicle are clearlydifferent. Thus it is known that the keratins of the hair shaftrepresent a family (Langbein et al., 2001, J. Biol. Chem. 276:35123-35132) distinct from that expressed in the epidermis, thatdifferentiation markers such as keratins K1 and K10 are not expressed inthe hair follicle and in particular in the outer sheath (Lenoir et al.,1988, Dev. Biol. 130: 610-620), that trichohyalin (O'Guin et al., 1992,J. Invest. Dermatol. 98: 24-32) and keratin K6irs (Porter et al., 2001,Br. J. Dermatol. 145: 558-568) are expressed in the hair follicle, inparticular in the inner sheath but not in the epidermis, and thatcyclooxygenase type 1, while being expressed in the epidermis, is not inthe keratinocytes of the hair follicle but in the dermal papilla(Michelet. et al., 1997, J. Invest. Dermatol. 108:205-209).

Surprisingly, the applicant has now demonstrated that an enzyme which isspecifically involved in the degradation of these prostaglandins ispresent in the dermal papilla of the hair, which is a criticalcompartment for the life of the hair. Indeed, the applicant has nowproved the presence of 15-hydroxyprostaglandin dehydrogenase (15-PGDH)at this level. It has additionally shown that the specific inhibition of15-PGDH has a beneficial effect on the hair density and/or growth.

Accordingly, the subject of the present invention is a composition, inparticular a cosmetic composition, containing at least one15-hydroxyprostaglandin dehydrogenase inhibitor and excipients which arephysiologically acceptable, in particular from a cosmetic point of view.

The subject of the invention is thus the cosmetic use of at least one15-PGDH inhibitor in or for the preparation of a composition intendedfor increasing the density of keratinous fibres and in particular hair,and/or reducing the heterogeneity of their diameter and/or promotingtheir growth.

The subject of the invention is also the cosmetic use of at least one15-PGDH inhibitor in a care or make-Lip cosmetic composition for theeyelashes of human beings or for the preparation of a care and/ortreatment composition for the eyelashes of human beings, for or intendedfor inducing and/or stimulating the growth of the eyelashes and/orincreasing their density. This composition thus makes it possible tomaintain the eyelashes in a good state and/or to improve theirappearance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the expression of 15-PGDH in the hair follicles fromcultures of fibroblasts of dermal papillae of human hair in agarose geltinder ultraviolet light, with a characteristic MW band shown at 706 bp;

FIG. 2 illustrates the expression of prostaglandin synthase in the hairfollicles from cultures of fibroblasts of dermal papillae of human hairin agarose gel under ultraviolet light, with a characteristic 1061 pb MWband shown; and

FIG. 3 depicts the results of electrophoretic analyses (SDS-page) ofpurified 15-PGDH and PGFS.

15-PGDH is a key enzyme in the deactivation of prostaglandins, inparticular of PGF2α, and of PGE2, which are important mediators of hairgrowth and survival. 15-PGDH type 1 corresponds to the EC 1.1.1.141classification and is NAD+-dependent. It was isolated from pig kidney,its inhibition by a thyroid hormone, triiodothyronine, at doses whichare much higher than physiological doses, has in particular beenobserved 15-PGDH type 2 is NADP-dependent.

In the text which follows, the expression “15-PGDH” can denote eitherform of the enzyme, or both.

However, the presence of 15-PGDH in the dermal papilla has never beendemonstrated, and it has never been proposed to use a 15-PGDH inhibitorfor maintaining and/or increasing hair density and/or for reducing theheterogeneity of the hair diameters in humans.

The expression “15-PGDH inhibitor” is understood to mean, for thepurposes of the invention, any substance, simple or complex compound, ofnatural or synthetic origin, capable of inhibiting or reducing theactivity of the 15-PGDH enzyme, and/or capable of inhibiting, reducingor slowing the reaction catalysed by this enzyme.

Advantageously, the inhibitor is an inhibitor specific for 15-PGDH type1.

The expression “heterogeneity of the hair diameters” is understood tomean a wide variation in the diameters of the hair in the same region ofthe scalp, some hair strands having a physiological diameter of close to100 μm, others having, in the immediate vicinity of these hair strands,a reduced diameter (thin hair). Thus, the expression “reducing theheterogeneity of the diameters” is understood to mean increasing thediameter of thin hair.

The expression increasing the hair density is understood to meanincreasing the number of hair strands per cm³ of scalp.

Advantageously, a composition according to the invention will compriseexcipients suitable for administration to the targeted skin area orkeratinous fibre, and in particular the scalp, the hair or theeyelashes. The medium in which the 15-PGDH inhibitor is presentaccording to the invention may be anhydrous or aqueous. The compositionwill comprise for example a cosmetologically acceptable medium which mayconsist of water or of at least one solvent chosen from hydrophilicorganic solvents, lipophilic organic solvents, amphiphilic organicsolvents and mixtures thereof, in particular a mixture of water and atleast one of the abovementioned solvents.

For topical application, the composition which can be used according tothe invention may be in particular in the form of an aqueous,aqueous-alcoholic or oily solution or of a dispersion of the lotion orserum type, of emulsions of liquid or semiliquid consistency of the milktype, which are obtained by dispersing a fatty phase in an aqueous phase(O/W) or conversely (W/O) or multiple emulsions, of a loose or compactpowder to be used as it is or to be incorporated into a physiologicallyacceptable medium, or of suspensions or emulsions of a soft consistencyof the aqueous or anhydrous cream or gel type, or alternatively ofmicrocapsules or microparticles, or of vesicular dispersions of theionic and/or nonionic type. It may also be provided in the form of asalve, a tincture, a cream, an ointment, a powder, a patch, animpregnated pad, a solution, an emulsion or a vesicular dispersion, alotion, a gel, a spray, a suspension, a shampoo, an aerosol or a foam.It may be anhydrous or aqueous. It may also consist of solidpreparations constituting cleansing soaps or cakes.

These compositions are prepared according to the customary methods.

The composition which can be used according to the invention may inparticular consist of a composition for hair care, and in particular ashampoo or an after-shampoo, in particular for twice-weekly or weeklyapplication, a hair shaping lotion, a treatment lotion, a hair carelotion, for example for daily or twice-weekly application, a hairstyling cream or gel, restructuring lotions for hair, a mask, and thelike.

The cosmetic composition according to the invention will be preferably acream, a hair lotion, a shampoo or an after-shampoo, hair mascara ormascara for the eyelashes.

For application to the eyelashes or body hair, the composition to whichthe invention applies may be provided in the form of a mascara,pigmented or otherwise, to be applied with a brush or with a comb to theeyelashes, the eyebrows, the hair, or to beard or moustache hair.

For a composition for use by injection, the composition may be providedin the form of an aqueous lotion or an oily suspension. For use orally,the composition may be provided in the form of capsules, granules,syrups to be taken orally or tablets.

The quantities of the various constituents of the compositions which canbe used according to the invention are those conventionally used in thefields considered.

The aqueous phase contains water and optionally an ingredient which ismiscible in any proportion with water such as C₁ to C₈ lower alcoholssuch as ethanol, isopropanol, polyols such as propylene glycol,glycerol, sorbitol, or acetone or ether.

When the composition which can be used according to the invention is anemulsion, the proportion of the fatty phase may range from 2 to 80%, inparticular from 5% to 80% by weight, and preferably from 5% to 50% byweight relative to the total weight of the composition. The oils, waxes,emulsifiers and coemulsifiers used in the composition in the form of anemulsion are chosen from those conventionally used in the cosmeticfield. The emulsifier and the coemulsifier are present in thecomposition in a proportion ranging from 0.1, in particular from 0.3% to30% by weight, preferably from 0.5 to 20% by weight, and even betterfrom 1 to 8% relative to the total weight of the composition. Theemulsion may additionally contain lipid vesicles and in particularliposomes.

When the composition which can be used according to the invention is anoily solution or gel, the fatty phase may represent more than 90% of thetotal weight of the composition.

Advantageously, the composition will comprise 10 microspheres,nanospheres, liposomes, oleosomes or nanocapsules, into which at leastone 15-PGDH inhibiting agent will be encapsulated Examples of suchformulations are described in particular in patents EP 199636, EP375520, EP 447318, EP 557489, WO 97/12602, EP 1151741 or U.S. Pat. No.5,914,126.

By way of example, the microspheres may be prepared according to themethod described in patent application EP 0 375 520.

The nanospheres may be provided in the form 20 of an aqueous suspensionand may be prepared according to the methods described in patentapplications FR 0015686 and FR 0101438.

The oleosomes consist of an oil-in-water emulsion formed by oilyglobules provided with a lamellar liquid crystal coating dispersed in anaqueous phase (see patent applications EP 0 641 557 and EP 0 705 593).

The 15-PGDH inhibitor may also be encapsulated into nanocapsulesconsisting of a lamellar coating obtained from a silicone surfactant asdescribed in patent application EP 0 780 115, the nanocapsules may alsobe prepared based on water-dispersible sulphonic polyesters for exampleaccording to the technique described in patent application FR 0113337.

Advantageously, for a hair application, the composition is an aqueous,alcoholic or aqueous-alcoholic solution or suspension, and even better awater/ethanol solution or suspension. The alcoholic fraction mayrepresent from 5% to 99.9% and even better from 8% to 80%.

For a mascara application, the composition is a wax-in water orwax-in-oil dispersion, a gelled oil, an aqueous gel, pigmented orotherwise.

In a known manner, the composition according to the invention may alsocontain adjuvants which are customary in the cosmetic field, such ashydrophilic or lipophilic gelling agents or thickeners, hydrophilic orlipophilic additives, preservatives, antioxidants, solvents, perfumes,fillers, screening agents, odour absorbers, electrolytes, neutralizers,UV blocking agents such as sun-screening agents, film-forming polymers,cosmetic and pharmaceutical active agents with a beneficial action onthe skin or the keratinous fibres and dye substances, which are solubleor insoluble in the medium. The quantities of these various adjuvantsare those conventionally used in the cosmetic field, and in particularare from 0.01 to 50% of the total weight of the composition, for examplefrom 0.01% to 20%, in particular less than or equal to 10% of the totalweight of the composition, and in particular greater than or equal to0.1%. These adjuvants, depending on their nature, may be introduced intothe fatty phase, into the aqueous phase and/or into the lipid spherules,vesicles or microspheres, such as liposomes.

The fatty phase may contain fatty or oily compounds, which are liquid atroom temperature (25° C.) and atmospheric pressure (760 mmHg), generallycalled oils. These oils may be compatible or otherwise with each otherand may form a macroscopically homogeneous liquid fatty phase or a two-or three-phase system.

The fatty phase may, in addition to the oils, contain waxes, gums,lipophilic polymers, “pasty” or viscous products containing solid partsand liquid parts.

As oils or waxes which can be used in the invention, there may bementioned mineral oils (petroleum jelly, hydrogenated isoparaffin),vegetable oils (liquid fraction of shea butter, sunflower oil, soya-beanoil, wheat oil), animal oils (perhydrosqualene), synthetic oils(Purcellin oil, fatty acid esters), silicone oils or waxes (linear orcyclic polydimethylsiloxanes, cyclomethicone, phenyltrimethicone) andfluorinated oils (perfluoropolyethers), beeswax, candelilla wax,carnauba wax or paraffin wax. It is also possible to add fatty alcoholsand free fatty acids (stearic, linoleic or linolenic acid) to these oilsand waxes.

As emulsifiers which can be used in the invention, there may bementioned for example glyceryl stearate or laurate, sorbitol stearatesor oleates, alkyl dimethiconecopolyol (with alkyl ≧8) and mixturesthereof, polyoxyethylenated sorbitol stearate or oleate, for examplepolysorbate 60 and the PEG-6/PEG-32/glycol stearate mixture sold underthe name Tefose® by the company Gattefosse, polyethylene glycolmonostearate or monolaurate, dimethicone copolyols and mixtures thereof.

As solvents which can be used in the invention, there may be mentionedlower alcohols, in particular ethanol and isopropanol, and propyleneglycol.

As hydrophilic gelling agents which can be used in the invention, theremay be mentioned carboxyvinyl polymers (carbomer), acrylic copolymerssuch as acrylate/alkyl acrylate copolymers, polyacrylamides,polysaccharides such as hydroxypropylcellulose, natural gums and clays,and, as lipophilic gelling agents, there may be mentioned modified clayssuch as Bentones®, metal salts of fatty acids such as aluminiumstearates and hydrophobic silica, ethyl cellulose, and polyethylene.

Preferably, the inhibiting agent(s) are present at a concentrationgreater than or equal to 10⁻³%, in particular of 0.001% to 5% w/vrelative to the composition, more preferably of 0.01 to 2%. However,these quantities will be adjusted by persons skilled in the artaccording to the compound used, in order to obtain an optimumanti-hair-loss activity and an enzyme inhibition activity equivalent toa practically total inhibition of the 15-PGDH enzyme, under conditionsfor applying the composition, the concentration used being generallygreater than or equal to that for which a 100% inhibition of 15-PGDH isobserved in vitro. The concentration of 15-PGDH inhibiting agent will bein particular 50 to 500 times higher than the concentration for which100% inhibition of 15-PGDH was observed in vitro, for exampleconcentrations about 100 times that corresponding to a total inhibitionin vitro will be used.

Suitable 15-PGDH inhibitors may be determined by persons skilled in theart, the inhibiting agent will be chosen in particular from traxanox,its salts and its esters.

According to a particularly advantageous embodiment of the invention,the composition will comprise at least one inhibitor specific for15-PGDH, the expression “specific inhibitor” is understood to mean anactive agent which is scarcely or not inhibitory for the synthesis ofprostaglandins, in particular for the synthesis of PGF2α or of PGF2.Preferably, the 15-PGDH inhibitor will be scarcely or not inhibitory forprostaglandin synthase (PGF synthase).

Indeed, in the context of its research studies, the applicant has nowfound, unexpectedly, that PGF synthase is also expressed in the dermalpapilla. Maintaining an effective quantity of prostaglandins at the siteof action therefore results from a complex biological balance betweenthe synthesis and the degradation of these molecules. The exogenoussupply of compounds exhibiting catabolism will therefore be lesseffective if this activity is combined with an inhibition of synthesis.

As a supplement or as a replacement for this exogenous supply, theapplicant has now demonstrated that it is possible to promote themaintenance of an endogenous prostaglandin pool, and therefore themaintenance or even the increase in the density of the keratinousfibres, in particular of the hair density, and the maintenance of thequality of the keratinous fibres, in particular of hair.

As an application of the present invention, it is now possible to targetparticularly active compounds, for which the 15-PGDH inhibiting activityis significantly greater than the PGF synthase inhibiting activity. Theratio between the respectively 15-PGDH and PGF synthase inhibitingactivities for the administered dose, determined in particular by theconcentrations inhibiting 50% of the enzyme activity, will be at leastgreater than 17 preferably at least 3:1 advantageously greater than orequal to 5:1. Agents which are particularly suitable for carrying outthe invention have a ratio between the 15-PGDH and PGF synthaseinhibiting activities greater than or equal to 10:1 in particulargreater than or equal to 15, preferably greater than or equal to 25:1.

In this regard, compounds suitable for carrying out the invention arethe compounds corresponding to the following formulae.

Molecule A: 5-amino-4,6-dichloro-2-phenylpyrimidine

Molecule B:N-[7-(2-Chlorophenyl)-5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl]benzamide

The choice of other agents which are active in the inhibition of 15-PGDHaccording to the present invention will be made by persons skilled inthe art using a simple test starting with potential candidates. Thistest will consist in comparing the kinetics of a reaction catalysed bythis enzyme, in a reaction medium comprising a substrate for the enzymeand possible cosubstrates, in the presence or otherwise of the compoundwhose 15-PGDH inhibiting role it is desired to evaluate; the reactionconditions (pH, temperature, reaction time, and the like) are thosesuitable for the reaction and are the same for the measurement in thepresence or in the absence of the compound or substance to be tested.

For that, the following are for example brought into contact: the enzyme15-PGDH at a final concentration of 7×10⁻³ mg/ml, its cosubstrate(β-NAD) and a substrate (PGF2) at the concentrations corresponding tothe conditions conventionally used for this test as described forexample by Cho and Tai (Inhibition of NAD-dependent15-hydroxyprostaglandin dehydrogenase (15-PGDH) by cyclooxygenaseinhibitors and chemopreventive agents. Prostaglandins, Leukotrienes andEssential Fatty Acids, 2002, 67(6): 461-465), for example 1.5 mM β-NADand 50 μM prostaglandin E2. The rate of the reaction is measured at 37°C. for 1 minute. The same reaction is carried out, but adding the testcompound to the medium at the start of the reaction. The maximum enzymereaction rate per unit of time (Vmax) measured in the presence of thecompound is compared to that of the control with no compound, and thepercentage inhibition [100−(Vmax assay×100)/Vmax control] is determined.

The compounds noted as 15-PGDH inhibitors are then tested for theircapacity to inhibit PGF synthase. For that, the following are forexample brought into contact the enzyme PGFS at a final concentration of25×10⁻³ mg/l, its cosubstrate (β-NADPH2) and a substrate (for examplephenanthrenequinone) at the concentrations conventionally used for thistest as described for example by Suzuki et al. (cDNA cloning, expressionand mutagenesis study of liver-type prostaglandin F synthase. J BiolChem, 1999, 274(1): 241-248), that is to say 100 μM β-NADPH2 and 20 μMphenanthrenequinone. The maximum reaction rate is measured per unit oftime at 37° C. The same reaction is carried out but adding the testcompound to the medium at the start of the reaction. The maximum enzymereaction rate with the compound is compared to that of the control withno compound, and the percentage inhibition [100−(Vmax assay×100)/Vmaxcontrol] is determined.

The percentage inhibition of the reaction catalysed by 15-PGDH and thatof the reaction catalysed by PGFS is then compared. More precisely, theratio of the IC50 values of a compound in relation to PGFS and 15-PGDH(IC50 PGFS/IC50 15-PGDH) is established. The IC50 is the concentrationof the compound for which Vmax is reduced by 50%.

The activity of compounds showing a selective inhibition of 15-PGDH forthe purposes of the present invention may also be demonstrated bymeasuring the amount of prostaglandins in a cellular model mimicking theenzymatic environment of the hair papilla. This makes it possible toevaluate the efficacy of a selective 15-PGDH inhibitor on the protectionof prostaglandins in a complex biological system producing the differenttypes of enzyme involved in the metabolism of these molecules. Forexample, a culture of promonocytes is used, these being precursors ofmacrophages under certain conditions, a model which is very widespreadfor studying the metabolism of prostaglandins.

Indeed, the phorbol esters (10 nM PMA) cause within 24 h 00 min thedifferentiation of the promonocyte line U937 into macrophages, thisdifferentiation is accompanied by the induction of 15-PGDH, (Tong andTai, Biochim Biophys Acta, 2000, 1497: 61-68).

Moreover, stimulation of these macrophages by LPS (lipopolysaccharideextracted from the bacterial wall) induces (at 100 ng/ml) in 6 h 00 minPGHS-2 (or COX-2), an enzyme responsible (in the same way as COX-1) forthe synthesis of PGH2, a precursor (inter alia) via PGFS, of PGF2α(Arias-Negrete et al., 1995 Biochem Biophys Res Commun, 208(2),582-589).

In a 1st stage, macrophage precursors are cultured in a suitable medium,in the presence of a compound stimulating their differentiation and theinduction of 15-PGDH, and then the production of prostaglandins by thesecells is stimulated, for example by LPSs in the form of an extract or bypurified LPSs, this 2nd stage is performed in the presence or otherwiseof the compound to be tested. The concentrations of prostaglandins, inparticular of PGF2α, obtained in the presence of the 15-PGDH inhibitingcompound to be tested are compared to that of the control containingonly the 15-PGDH inducer, it being possible to carry out thismeasurement by any method known to a person skilled in the art, inparticular by immunoenzymatic assay. At the time of the assay, thequantity of PGF2α measured is therefore the resultant of the 2 enzymaticactivities for which the compounds tested are more or less active: thatof PGFS which leads to the synthesis of PCF2α and that of 15-PGDH whichleads to the degradation. In the presence of a nonselective 15-PGDHinhibitor (also inhibitor of PGFS), a reduction in PGF2α will beobserved (corresponding to a reduction in synthesis by the action of theproduct on PGFS). In the presence of an inhibitor selective for 15-PGDH,an increase in PGF2α will be observed, which corresponds to a reductionin degradation. The compounds for which the PGF2α level observed isgreater by at least 5%, preferably by at least 10% than that of thecontrol (inducer of the synthesis of prostaglandin alone) therefore havea prostaglandin F2α protecting role. Advantageously, for the compoundssuitable for carrying out the invention, the prostaglandin concentrationwith the compound to be tested is equal to or greater by at least 20%,or even by 30% than that of the control.

Such compounds are in particular certain salified or nonsalifiedacetyltetrazoles, endowed with a surprising activity favourable toimproving the hair density. The applicant has indeed found that thesecompounds are 15-hydroxyprostaglandin dehydrogenase inhibitors.

Another subject of the invention is therefore a hair care compositioncontaining, in a physiologically acceptable medium, an effectivequantity of a tetrazole compound of formula (I) or (II) or of one of itssalts.

in which:

R₁ and R₂ are independently chosen from hydrogen, halogen, OR₅, SR₅,NR₅R′₅, COOR₅, COR₅, CONR₅R′₅, CF₃, CN, NR₅COR′₅, SO₂R₅, SO₂NR₅R′₅,NR₅SO₂R′₅, CSR₅, OCOR₅, COSR₅, SCOR₅, CSNR₅R′₅, NR₅CONR′₅R″₅,NR₅C(═NR′₅)NR″₅R′″₅, NR₅CSNR′₅, NR₅CSNR′₅R′″₅, linear or branched C₁-C₂₀alkyl radicals, and 4- to 7-atom rings, optionally containing at leastone heteroatom, it being possible for these rings to be separated orfused, the alkyl radicals and the rings being additionally saturated orunsaturated, and optionally substituted with at least one substituentA₁, where R₅, R′₅, R″₅ and R′″₅ independently denote hydrogen, a linearor branched C₁-C₂₀ alkyl radical or a 4- to 7-atom hydrocarbon ring, thehydrocarbon ring or the alkyl radical being saturated or unsaturated andoptionally substituted with at least one substituent A₂;

R₃ is chosen from hydrogen, OR₆, SR₆, NR₆R′₆, CF₃, NR₆COR′₆, NR₆SO₂R′₆,NR₆CONR′₆R″₆, NR₆CSR′₆, NR₆CSNR′₆R″₆, linear or branched C₁-C₂₀ alkylradicals, and separated or fused 4- to 7-atom hydrocarbon rings, thealkyl radicals and the hydrocarbon rings being additionally saturated orunsaturated and optionally substituted with at least one substituent A₃,with R₆, R′₆ and R″₆ independently denoting hydrogen, a linear orbranched C₁-C₂₀ alkyl radical, or a 4- to 7-atom hydrocarbon ring, thealkyl radical or the hydrocarbon ring being saturated or unsaturated andoptionally substituted with at least one substituent A₄;

R₄ is chosen from hydrogen, COOR₇, CONR₇R′₇, SO₂R₇, SO₂NR₇R′₇, COR₇,CSR₇, COSR₇, CSNR₇R′₇, linear or branched C₁-C₂₀ alkyl radicals andseparated or fused 4- to 7-atom hydrocarbon rings, the alkyl radicalsand hydrocarbon rings being additionally saturated or unsaturated andoptionally substituted with at least one substituent A₅; R₄ mayadditionally represent, in the case of formula (II), a halogen, OR₇,SR₇, NR₇R′₇, CF₃, CN, NR₇COR′₇, NR₇SO₂R′₇, OCOR₇, SCOR₇, NR₇CONR′₇R″₇,NR₇C(═NR′₇)NR″₇R′″₇, NR₇CSR′₇ or NR₇CSNR′₇R″₇, with R₇, R′₇, R″₇ andR′″₇ independently denoting hydrogen, a linear or branched C₁-C₂₀ alkylradical, or a 4- to 7-atom hydrocarbon ring, the alkyl radical or thehydrocarbon ring being saturated or unsaturated and optionallysubstituted with at least one substituent A₆;

A₁ and A₂ are independently chosen from halogens, heterocyclescontaining from 4 to 7 atoms and at least one heteroatom, OR₈, SR₈,NR₈R′₈, COOR₈, CONR₈R′₈, CF₃, CN, NR₈COR′₈, SO₂R₈, SO₂NR₈R′₈, NR₈SO₂R′₈,COR₈, CSR₈, OCOR₈, COSR₈, SCOR₈, CSNR₈R′₈, NR₈CONR′₈R″₈,NR₈C(═NR′₈)NR″₈R′″₈, NR₈CSR′₈, NR₈CSNR′₈R″₈;

A₃ and A₄ are independently chosen from halogens, R₉, OR₉, SR₉, NR₉R′₉,COOR₉, CONR₉R′₉, CF₃, CN, NR₉COR′₉, SO₂R₉, SO₂NR₉R′₉, NR₉SO₂R′₉, CSR₉,OCOR₉, COSR₉, SCOR₉, CSNR₉R′₉, NR₉CONR′₉R″₉, NR₉C(═NR′₉)NR″₉R′″₉,NR₉CSR′₉, NR₉CSNR′₉R″₉;

A₅ and A₆ are independently chosen from halogens, R₁₀, OR₁₀, SR₁₀,NR₁₀R′₁₀, CF₃, CN, NR₁₀COR′₁₀, SO₂R₁₀, SO₂NR₁₀R′₁₀, NR₁₀SO₂R′₁₀, CSR₁₀,OCOR₁₀, SCOR₁₀, CSNR₁₀R′₁₀, NR₁₀CONR′₁₀R″₁₀, NR₁₀C(═NR′₁₀)NR″₁₀R′″₁₀,NR₁₀CSR′₁₀, NR₁₀CSNR′₁₀R′₁₀;

R₈, R′₈, R″₈, R′″₈, R₉, R′₉, R″₉, R′″₉, R₁₀, R′₁₀, R″₁₀ and R′″₁₀independently denoting hydrogen, a saturated or unsaturated, linear orbranched C₁-C₂₀ alkyl radical, a saturated or unsaturated 4- to 7-atomhydrocarbon ring, or a benzyl radical.

The invention also relates to the use of at least one tetrazole compoundof formula (I) or (II) or tautomeric form or of one of its salts, asdefined above, as agent for inducing and/or stimulating the growth ofthe hair of human beings and/or slowing its loss and/or increasing itsdensity.

The invention also relates to the cosmetic use of at least one tetrazolecompound of formula (I) or (II) or of one of its salts in a hair carecosmetic composition for human beings for reducing the loss of hairand/or increasing its density. Its subject is also the use of at leastone tetrazole compound of formula (I) or of one of its salts forpreparing a hair composition for human beings, intended for inducingand/or stimulating the growth of hair and/or slowing its loss and/orincreasing its density.

In particular, the invention also relates to the cosmetic use of atleast one tetrazole compound of formula (I) or (II) or of one of itssalts in a hair care cosmetic composition for human beings or forpreparing a hair composition for human beings intended for and/or fortreating androgenic alopecia. Thus, this composition makes it possibleto maintain the hair in a good state and/or to control the natural lossof hair in men.

The subject of the invention is also the use of at least one tetrazolecompound of formula (I) or (II) or of one of its salts as a15-hydroxyprostaglandin dehydrogenase inhibitor. Its subject is also theuse of at least one tetrazole compound of formula (I) or (II) or of oneof its salts for the manufacture of a composition intended for treatingdisorders linked to 15-hydroxyprostaglandin dehydrogenase in humanbeings.

Advantageously, the compounds of formula (I) or (II), in salified ornonsalified form, exhibit a 15-PGDH inhibiting activity which is greaterthan the PGF synthase inhibiting activity.

In the text which follows, and unless otherwise stated, the use of theterm “compound of formula (I) or (II)” should be understood to mean boththe compound of formula (I) or of formula (II) in acid or base form, andin salt form.

“At least one” according to the invention means one or more (2, 3 ormore). In particular, the composition may contain one or more compoundsof formula (I), one or more compounds of formula (II) or a mixture ofcompounds of formula (I) and of formula (II). This or these compoundsmay be cis or trans isomers or a mixture of cis/trans isomers. They mayalso be in tautomeric form. They may also be enantiomers and/ordiastereoisomers or a mixture of these isomers, in particular a racemicmixture.

The expression “hydrocarbon” ring is understood, for the purposes of theinvention, to mean a ring containing only carbon-carbon bonds to formthe ring.

According to the invention, the rings used for R₁ to R₄ in the formulae(I) and (II) independently contain from 4 to 7 atoms and even betterfrom 5 to 6 atoms. They may be saturated or unsaturated. They mayfurthermore be alone or fused with another ring of the same chemicalstructure or otherwise. In addition, R₁ and R₂ optionally contain one ormore heteroatoms such as S, N, O or combinations thereof.

According to the invention, the rings used for R₅, R′₅, R″₅, R′″₅, R₆,R′₆, R″₆, R′″₆, R₇, R′₇, R″₇, R′″₇, R₈, R′₈, R″₈, R′″₈, R₉, R′₉, R″₉,R′″₉, R₁₀, R′₁₀, R″₁₀ and R′″₁₀ in the formulae (I) and (II)independently contain from 4 to 7 carbon atoms and even better from 5 to6 carbon atoms. They may be saturated or even better unsaturated.

Moreover, the heterocycles used for A₁ and A₂ in the formulae (I) and(II) contain one or more heteroatoms such as S, N, O or combinationsthereof. They additionally independently contain from 4 to 7 atoms andeven better from 5 to 6 atoms. Furthermore, they may be saturated orunsaturated.

As saturated hydrocarbon rings which can be used in the formula (I) or(II), the cyclopentyl or cyclohexyl radical may be mentioned. Asheterocycle, there may be mentioned the pyridine, piperidine,morpholine, pyrrole, furan and thiazole rings. As unsaturatedhydrocarbon rings, the phenyl or naphthyl radical may be mentioned. Inaddition, these rings may be substituted with one or more substituentshaving the meaning indicated above for A₁, A₂, A₃, A₄, A₅ and A₆,depending on whether R₁, R₂, R₃, R₄, R₅, R′₅, R″₅, R′″₅, R₆, R′₆, R″₆,R₇, R′₇, R″₇ or R′″₇ is involved.

The expression “linear or branched C₁-C₂₀ alkyl radical” is understoodto mean, according to the invention, the acyclic radicals obtained fromthe removal of a hydrogen atom from the molecule of a linear or branchedhydrocarbon having from 1 to 20 carbon atoms, and in particular themethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,hexyl, heptyl and octyl radicals, and their corresponding positionisomers. As example of (saturated) alkyl radical which can be used inthe invention, there may be mentioned the methyl, ethyl, n-butyl,isopropyl and n-hexyl radicals.

As halogen atom, the chlorine, fluorine, iodine or bromine, and moreespecially chlorine, atoms may be used.

According to the invention, the compounds of formula (I) or (II) are inisolated, that is to say nonpolymeric, form. In addition, thesubstituents A₁, A₂, A₃, A₄, A₅ and A₆ may be situated at any positionof the ring carrying them, and in particular at the position adjacent tothe group carrying the tetrazole ring.

According to one embodiment, at least one of R₁ and R₂ represent ahydrogen atom, a halogen atom and in particular a fluorine or chlorineatom. In particular, R₁ and R₂ represent hydrogen.

Advantageously, R₃ represents NR₆R′₆ or an aryl radical, and in oneparticular embodiment a naphthyl or phenyl radical, optionallysubstituted with the substituent A₃. In particular, A₃ represents OR₉.

According to one embodiment, R₆ represents hydrogen and R′₆ an aryl, inparticular a phenyl, radical optionally substituted with the group OR₉.

In particular, R₉ represents a saturated, linear or branched, C₁-C₂₀,and even better C₁-C₁₀, alkyl radical and for example the methylradical.

According to one embodiment of the invention, R₄ represents an arylradical and in particular a naphthyl or phenyl radical.

The expression “salts of the compound of formula (I) or (II)” isunderstood to mean, according to the invention, the organic or inorganicsalts of a compound of formula (I) or (II).

As inorganic salts which can be used according to the invention, theremay be mentioned the sodium or potassium salts, and the zinc (Zn²⁺),calcium (Ca²⁺), copper (Cu²⁺), iron (Fe²⁺), strontium (Sr²⁺), magnesium(Mg²⁺) and manganese (Mn²⁺) salts; the hydroxides and the carbonates.

The organic salts which can be used according to the invention are forexample the triethanolamine, monoethanolamine, diethanolamine,hexadecylamine, N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine andtris-hydroxymethylaminomethane salts.

Compounds suitable for carrying out the invention are in particular the15-PGDH inhibiting compounds of formula (I) or (II) as defined above, inwhich:

R₁ and R₂ are independently chosen from hydrogen, halogens, OR₅, SR₅,NR₅R′₅, COOR₅, CF₃, CN, linear or branched C₁-C₂₀ alkyl radicals, and 4-to 7-atom rings, optionally containing at least one heteroatom, it beingpossible for these rings to be separated or fused, the alkyl radicalsand the rings being additionally saturated or unsaturated, where R₅ andR′₅ independently denote hydrogen, a linear or branched C₁-C₂₀ alkylradical, or a 4- to 7-atom hydrocarbon ring, the hydrocarbon ring or thealkyl radical being saturated or unsaturated;

R₃ is chosen from hydrogen, OR₆, SR₆, NR₆R′₆, CF₃, linear or branchedC₁-C₂₀ alkyl radicals, and separated or fused 4- to 7-atom hydrocarbonrings, the alkyl radicals and the hydrocarbon rings being additionallysaturated or unsaturated and optionally substituted with at least onesubstituent A₃, with R₆ and R′₆ independently denoting hydrogen, alinear or branched C₁-C₂₀ alkyl radical, or a 4- to 7-atom hydrocarbonring, the alkyl radical or the hydrocarbon ring being saturated orunsaturated and optionally substituted with at least one substituent A₄;

R₄ is chosen from hydrogen, COOR₇, CSR₇ linear or branched C₁-C₂₀ alkylradicals, and separated or fused 4- to 7-atom hydrocarbon rings, thealkyl radicals and the hydrocarbon rings being additionally saturated orunsaturated, R₄ may additionally represent, in the case of formula (II),a halogen, OR₇, SR₇, NR₇R′₇, CF₃, CN, with R₇ and R′₇ independentlydenoting hydrogen, a linear or branched C₁-C₂₀ alkyl radical, or a 4- to7-atom hydrocarbon ring, the alkyl radical or the hydrocarbon ring beingsaturated or unsaturated, optionally substituted with at least onesubstituent chosen from OR₁₀, SR₁₀, NR₁₀R′₁₀, CF₃, with R₁₀ and R′₁₀denoting a saturated or unsaturated, linear or branched C₁-C₂₀ alkylradical;

A₃ and A₄ are independently chosen from halogens, R₉, OR₉, SR₉, NR₉R′₉,COOR₉, CF₃, with R₉ and R′₉ independently denoting hydrogen, a saturatedor unsaturated, linear or branched C₁-C₂₀ alkyl radical, a saturated orunsaturated 4 to 7-atom hydrocarbon ring, or a benzyl radical.

According to another embodiment of the invention, the 15-PGDH inhibitingcompounds are such that in formula (I) or (II) defined above,

R₃ is chosen from hydrogen, OR₆, SR₆, NR₆R′₆, CF₃, linear or branchedC₁-C₂₀ alkyl radicals, and separated or fused 4 to 7-atom hydrocarbonrings, the alkyl radicals and the hydrocarbon rings being additionallysaturated or unsaturated and optionally substituted with at least onesubstituent A₃, with R₆ and R′₆ independently denoting hydrogen, alinear or branched C₁-C₂₀ alkyl radical, or a 4- to 7-atom hydrocarbonring, the alkyl radical or the hydrocarbon ring being saturated orunsaturated and optionally substituted with at least one substituent A₄;

R₄ is chosen from hydrogen, linear or branched C₁-C₂₀ alkyl radicals,and separated or fused 4- to 7-atom hydrocarbon rings, the alkylradicals and the hydrocarbon rings being additionally saturated orunsaturated, optionally substituted with at least one suibstituentchosen from OR₁₀, SR₁₀, NR₁₀R′₁₀, CF₃, with R₁₀ and R′₁₀ denoting asaturated or unsaturated, linear or branched C₁-C₂₀ alkyl radical;

A₃ and A₄ are independently chosen from R₉, OR₉, SR₉, NR₉R′₉, COOR₉,with R₉ and R′₉ independently denoting hydrogen, a saturated orunsaturated, linear or branched C₁-C₂₀ alkyl radical, a saturated orunsaturated 4- to 7-atom hydrocarbon ring, or a benzyl radical.

As examples of tetrazole compounds of formula (I) which can be used inthe invention, the following compounds may be mentioned,

-   Compound 1: 1-phenyl-2-(2-phenyl-2H-tetrazol-5-yl)ethanone

-   Compound 2:    1-(2-methoxyphenyl)-2-(2-phenyl-2H-tetrazol-5-yl)ethanone

-   ethyl 2-(2-phenyl-2H-tetrazol-5-yl)acetate

-   2-(2-phenyl-2H-tetrazol-5-yl)acetic acid

-   1-(3,4,5-trimethoxyphenyl)-2-(2-phenyl-2H-tetrazol-5-yl)ethanone

-   4-[2-(2-phenyl-2H-tetrazol-5-yl)acetyl]benzoic acid

-   1-(4-benzyloxyphenyl)-2-(2-phenyl-2H-tetrazol-5-yl)ethanone

-   3-methoxy-1-phenyl-2-(2-phenyl-2H-tetrazol-5-yl)propan-1-one

As examples of tetrazole compounds of formula (II) which can be used inthe invention, the following compounds may be mentioned.

-   Compound 3. N-(2-phenyl)-2-(5-phenyl-2H-tetrazol-2-yl)acetamide

-   Compound 4:    N-(2-methoxyphenyl)-2-(5-phenyl-2H-tetrazol-2-yl)acetamide

-   N-(4-methylphenyl)-2-{5-[3-(trifluoromethyl)phenyl]-2H-tetrazol-2-yl}acetamide.

-   N-(2-methoxyphenyl)-2-(2H-tetrazol-2-yl)acetamide

-   2-(5-phenyl-2H-tetrazol-2-yl)acetamide

-   N-(2-methoxyphenyl)-2-[5-(2-naphthyl)-2H-tetrazol-2-yl]acetamide

-   N-(2-methoxyphenyl)-2-[5-(4-methoxyphenyl)-2H-tetrazol-2-yl)acetamide

-   N-(3-hydroxypropyl)-2-(5-phenyl-2H-tetrazol-2-yl)acetamide

-   3-hydroxy-N-(2-methoxyphenyl)-2-(5-phenyltetrazol-2-yl)propionamide

-   3-methoxy-N-(2-methoxyphenyl)-2-(5-phenyltetrazol-2-yl)propionamide

The compounds of formula (I) or (II), salified or otherwise, may bemanufactured in a known manner

1) Preparation of 5-acetyltetrazoles (Formula I)

The compounds of formula (I) of the invention may be prepared by amethod described in the literature: D. Moderhack et al., J. Chem. Soc.Perkin Trans. 1, 2001, 720-728. The reaction scheme is the following:

2) Preparation of 2-acetyltetrazoles (Formula II)

The compounds of formula (II) of the 10 invention may be prepared byalkylation, with α-chlorocarbonyl-containing reagents, of tetrazolessubstituted at the 5-position. This reaction is particularly suitable inthe case of the synthesis of 5-phenyltetrazoles (corresponding toR₄=phenyl). This type of preparation is known to persons skilled in theart and in particular from the document F. Eindberg, J. Org. Chem.,1970, 35, 11, 3978-3980.

The reaction scheme may be the following;

To the knowledge of the applicant, no prior art document describes orsuggests that the tetrazole compounds of formula (I) or (II) or theirsalts have properties of inducing and/or stimulating the growth of hairand/or of slowing its loss or that these compounds may be used topicallyto increase hair density.

The effective quantity of a compound of formula (I) or (II) or of one ofits salts corresponds to the quantity necessary to obtain the desiredresult (namely to increase the hair density). Persons skilled in the artare therefore able to evaluate this effective quantity which depends onthe nature of the compound used, on the person to which it is applied,and on the time of this application.

In the text which follows, unless otherwise stated, the quantities ofthe various ingredients of the composition are given as the percentageby weight relative to the total weight of the composition.

To give an order of magnitude, according to the invention, the compoundof formula (I) or one of its salts may be used in a quantityrepresenting from

10⁻³% to 5% of the total weight of the composition, and preferably in aquantity representing from 10⁻²% to 2% of the total weight of thecomposition, for example from 0.5 to 2%.

The composition of the invention may be for cosmetic or pharmaceuticaluse. Preferably, the composition of the invention is for cosmetic use.Accordingly, the composition should contain a physiologically acceptablemedium which is nontoxic and capable of being applied to the skin, thesuperficial body growths or the lips of human beings. The expression“cosmetic” is understood to mean, for the purposes of the invention, acomposition having a pleasant appearance, odour and feel.

The compound of formula (I) or (II), salified or otherwise, may be usedin a composition which has to be ingested, injected or applied to theskin (over any skin area to be treated).

According to the invention, the compound of formula (I) or (II) may beused orally in a quantity of 0.1 to 300 mg per day, for example of 5 to10 mg/d.

A preferred composition of the invention is a composition for cosmeticuse and in particular for topical application to the skin, and moreespecially to the scalp.

According to an advantageous embodiment, the compositions according tothe invention additionally comprise at least one agent which isbeneficial to the hair, such as in particular silicones, vegetable,animal, mineral or synthetic oils, waxes, ceramides, pseudoceramides,cationic polymers, sunscreens and vitamins.

The silicones which can be used in accordance with the invention are inparticular polyorganosiloxanes which are insoluble in the compositionand may be provided in the form of oils, waxes, resins or gums.

The organopolysiloxanes are defined in greater detail in the book byWalter NOLL “Chemistry and Technology of Silicones” (1968) AcademiePress. They may be volatile or nonvolatile.

The 15-PGDH inhibiting agents will, according to one of the embodimentsof the invention, be combined with other active compounds in order topromote the regrowth and/or limit the loss of hair.

These compounds will be chosen in particular from lipoxygenaseinhibitors as described in EP 648488, the bradykinin inhibitorsdescribed in particular in EP 845700, prostaglandins and theirderivatives, in particular those described in WO 98/33497, WO 95/11003,JP 97-100091, JP 96-134242, the agonists or antagonists of the receptorsfor prostaglandins, the nonprostanoic analogues of prostaglandins asdescribed in EP 1175891 and EP 1175890, WO 01/74307, WO 01/74313, WO01/74314, WO 01/74315 or WO 01/72268.

Agents promoting hair growth which may be present in the compositionsaccording to the invention include vasodilators, antiandrogens,cyclosporins and their analogues, antimicrobials, anti-inflammatoryagents with the exception of the inhibitors selective for prostaglandinH synthase I or COX-1, triterpenes, alone or as a mixture.

The vasodilators such as the potassium channel agonists includingminoxidil and its derivatives, aminexil and the compounds described inU.S. Pat. Nos. 3,382,247, 5,756,092, 5,772,990, 5,760,043, 5,466,694,5,438,058, 4,973,474, chromakalin and diazoxide may thus be present inthe composition.

The antiandrogens which can be used include in particular 5α-reductaseinhibitors such as finasteride and the compounds described in U.S. Pat.No. 5,516,779, cyprosterone acetate, azelaic acid, its salts and itsderivatives, and the compounds described in U.S. Pat. No. 5,480,913,flutamide and the compounds described in U.S. Pat. Nos. 5,411,981,5,565,467 and 4,910,226.

The antimicrobial compounds may be chosen from selenium derivatives,ketoconazole, triclocarban, triclosan, zinc pyrithione, itraconazole,asiatic acid, hinokitiol, mipirocine, and the compounds described in EP680745, clinycine hydrochloride, benzoyl or benzyl peroxide andminocycline.

The anti-inflammatory agents may be chosen from inhibitors specific forCox-2 such as for example NS-398 and DuP-697 (B. Batistini et al., DN&P1994; 7(8):501-511) and/or inhibitors of lipoxygenases, in particular5-lipoxygenase, such as for example zileuton (F. J., Alvarez & R. T.Slade, Pharmaceutical Res. 1992, 9(11): 1465-1473).

Other active compounds for promoting the growth and/or limiting the lossof hair, which are present in the compositions may also be chosen fromthe group comprising aminexil and its derivatives,6-O-[(9Z,12Z)octadec-9,12-dienoyl]hexapyranose, as described in FR027293, FR 0212828, benzalkonium chloride, benzethonium chloride,phenol, oestradiol, chlorpheniramine maleate, chlorophyllin derivatives,cholesterol, cysteine, methionine, benzyl nicotinate, menthol,peppermint oil, calcium panthotenate panthenol, resorcinol, proteinkinase C inhibitors, prostaglandin H synthase 1 or COX-1 activators asdescribed in FR 2732597, glycosidase inhibitors, glycosaminoglycanaseinhibitors, pyroglutamic acid esters, hexosaccharidic oracylhexosaccharidic acids, substituted ethylenearyls, N-acylated aminoacids, flavonoids, derivatives and analogues of ascomycin, histamineantagonists, triterpenes such as ursolic acid and the compoundsdescribed in U.S. Pat. No. 5,529,769, U.S. Pat. No. 5,468,888, U.S. Pat.No. 5,631,282, saponins, proteoglycanase inhibitors agonists andantagonists of oestrogens, pseudopterins, cytokines and growth factorpromotors, IL-1 or IL-6 inhibitors, IL-10 promoters, TNF inhibitors,vitamins, such as vitamin D, analogues of vitamin B12 and panthotenol,hydroxy acids, benzophenones, esterified fatty acids as described in FR027293, FR 0212828 and hydantoin.

According to an advantageous embodiment of the invention, the 15-PGDHinhibiting agent will be combined with an active compound for promotingthe growth and/or limiting the loss of hair, which is capable of beingmetabolized by this enzyme. Indeed, by virtue of the work by theapplicant, it is now known that certain compounds conventionally usedfor promoting the growth and/or delaying or preventing the loss of hairhave a lower activity because of the presence of 15-PGDH throughreduction of the concentration and/or accelerated disappearance of thesubstances from the site of action. In accordance with the presentinvention, it is now possible to obtain compositions having an increasedefficacy, by combining an active compound for promoting the growthand/or delaying or preventing the loss of hair, which is capable ofbeing metabolized by 15-PGDH and a 15-PGDH inhibitor as defined above Asynergistic action of the active agents will thus be obtained forpromoting the growth and/or delaying or preventing the loss of hair inthese compositions.

The composition will additionally contain, for example, at least onecompound chosen from prostaglandins, in particular prostaglandin PGE1,PGE2, their salts, their esters, their analogues and their derivatives,in particular those described in WO 98/33497, WO 95/11003, JP 97-100091,JP 96-134242, in particular agonists of the prostaglandin receptors. Itmay in particular contain at least one compound such as the agonists (inacid form or in the form of a precursor, in particular in ester form) ofthe prostaglandin F2 alpha receptor (FP-R) such as for examplelatanoprost, fluprostenol, cloprostenol, bimatoprost, unoprostone, theagonists (and their precursors, in particular the esters such astravoprost) of the prostaglandin E2 receptors (FP1-R, FP2-R, FP3-R,EP4-R) such as 17-phenyl PGE2, viprostol, butaprost, misoprostol,sulprostone, 16,16-dimethyl PGE2, 11-deoxy PGE1, 1-deoxy PGE1, theagonists and their precursors, in particular esters, of theprostacycline (IP) receptor such as cicaprost, iloprost,isocarbacycline, beraprost, the agonists and their precursors, inparticular the esters, of the prostaglandin D2 receptor such as BW245C((4S)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidineheptanoicacid), BW246C((4R)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidineheptanoicacid), the agonists and their precursors, in particular the esters, ofthe receptor for the thromboxanes A2 (TP) such as I-BOP([1S-[1a,2a(Z),3b(1E,3S),4a]]-7-[3-[3-hydroxy-4-[4-(iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid).

Advantageously, the composition according to the invention will compriseat least one 15-PGDH inhibitor as defined above and at least oneprostaglandin or one prostaglandin derivative such as for example theprostaglandins of series 2 including in particular PGF2α and PGE2 insaline form or in the form of precursors, in particular of the esters(example isopropyl esters), their derivatives such as 16,16-dimethylPGE2, 17-phenyl PGE2 and 16,16-dimethyl PGF2α, 17-phenyl PGF2α,prostaglandins of series 1 such as 11-deoxyprostaglandin E1,1-deoxyprostaglandin E1 in saline or ester form, their analogues, inparticular latanoprost, travoprost, fluprostenol, unoprostone,bimatoprost, cloprostenol, viprostol, butaprost, misoprostol, theirsalts or their esters. Preferably, the composition contains at least oneFP receptor agonist as described for example in WO 03/009820 or aprostanoic or nonprostanoic agonist of the EP2 and/or EP4 receptors inparticular as described in EP 1175892.

The compositions according to the invention may also contain penetrationaccelerating agents. These compounds are known to persons skilled in theart and improve the passage of the agents to the site of action, theywill be conventionally present in the compositions at concentrationsgreater than or equal to 0.01%, in particular of 0.1 to 20% andpreferably of 0.1 to 5%. Compositions of this type which can be used arefor example urea and the compounds mentioned in application WO 01/74313.

According to another of its subjects, the invention relates to a methodof cosmetic treatment for promoting the growth and/or reducing,preventing or delaying the loss of hair, characterized in that theactivity and/or the expression of 15-PGDH by the hair follicle isinhibited or reduced, comprising the administration of at least one15-PGDH inhibitor. This method will be adapted in particular in order tomaintain or increase the hair density and to reduce the heterogeneity ofthe hair diameter, in physiological cases where these parameters aredisrupted, in a transient manner.

The administration of the 15-PGDH inhibiting agent(s) will be carriedout in particular in the form of a composition as defined above. Theadministration of the 15-PGDH inhibitor or of the composition containingit in the method according to the invention will be carried out by anyroute, in particular orally. However, the method is preferably carriedout by topical administration, in situ.

The 15-PGDH inhibitors used in the method according to the invention areas defined above, in particular, at least one specific 15-PGDHinhibitor, that is to say little or no PGFS inhibitor, will be used.

According to one of the variants of the method according to theinvention, a prostaglandin and/or a prostaglandin derivative will beadditionally administered, such as for example prostaglandins of series2 including in particular PGF2α and PGE2 in saline or ester form (forexample isopropyl esters), their derivatives such as 16,16-dimethylPGE2, 17-phenyl PGE2, 16,16-dimethyl PGF2α, 17-phenyl PGF2α,prostaglandins of series 1 such as 11-deoxyprostaglandin E1,1-deoxyprostaglandin E1 in saline or ester form their analogues, inparticular latanoprost, fluprostenol, unoprostone, bimatoprost,cloprostenol, viprostol, butaprost, misoprostol in saline or ester form,such as travoprost.

The 15-PGDH inhibiting agent(s) will be applied alone or in the form ofa mixture, and optionally in combination with other active agents forpromoting the growth and/or delaying or preventing the loss of hair, orpenetration accelerants as defined above.

It is also possible, for example, to apply the composition containing aneffective quantity of a 15-PGDH inhibiting compound, for example offormula (I) or (II), salified or otherwise, in the evening, to keep itin contact overnight and optionally to shampoo in the morning. Theseapplications may be repeated daily for one or more months depending onthe individual.

Thus, the subject of the present invention is also a method for thecosmetic treatment of hair and/or of the scalp, intended to stimulatethe growth of hair in human beings and/or to slow its loss,characterized in that it consists in applying to the hair and/or thescalp a cosmetic composition comprising an effective quantity of atleast one 15-PGDH inhibiting compound, for example of formula (I) or(II) or of one of its salts, in leaving it in contact with the hairand/or the scalp, and optionally in rinsing the hair and/or the scalp.

This method of treatment has the characteristics of a cosmetic method inso far as it makes it possible to improve the aesthetic quality of thehair by giving it greater vitality and an improved appearance. Inaddition, it can be used daily for several months.

Advantageously, in the method according to the invention, between 5 and500 μl of a solution or composition as defined above, comprising between0.001% and 5% of inhibitor, is administered.

According to yet another of its aspects, the subject of the invention isthe use of at least one 15-PGDH inhibitor as defined above, for thepreparation of a composition intended for reducing hair loss and/orpromoting hair regrowth in a mammal, and in particular in humans. Thesaid composition will additionally comprise physiologically acceptableexcipients, suitable for administration by the topical or systemicroute. The 15-PGDH inhibitors will be used in particular according tothe invention for the preparation of a composition, in particular apharmaceutical, dermatological or cosmetic composition, in combinationwith excipients known to persons skilled in the art. All the variants ofthe cosmetic compositions described in the preceding text can be adaptedfor the preparation of the said composition.

According to one of its embodiments, the compositions of the inventionare intended for preventing, reducing or slowing hair loss, especiallyfor the treatment of alopecia, in particular of androgenetic alopecia;androgenetic alopecia is distinct from autoimmune alopecia such asalopecia areata.

The following examples are intended to illustrate the invention withoutlimiting in any manner the scope thereof.

In these examples, reference will be made to the following figures:

FIG. 1: expression of 15-hydroxyPGDH in the hair follicles

FIG. 2: expression of prostaglandin synthase in the hair follicles

FIG. 3: electrophoretic analysis of purified 15-PGDH and PGFS

EXAMPLE 1 Demonstration of the Expression of the mRNA for 15-PGDH andPGFS in the Fibroblasts of Dermal Papillae of Hair in Culture 1.Dissection of the Hair Follicles

Hair follicles obtained from lifting from volunteer donors are dissectedaccording to the method described in the B. Bernard/O. Gaillard patentFR 2736721A1 of 17 Jan. 1997; U.S. Pat. No. 5,712,169A of 28 Jan. 1998.

The isolated follicles are placed in immersion in a Petri dishcontaining 20 ml of culture medium 199 Gibco reference 31153-018, LifeTechnologie, BP 96, Cergy Pontoise Cedex, supplemented with 1% (v/v) ofan antibiotic solution Gibco reference 15240-096.

Using two needles, reference N,N-2516R, Terumo Europe N.V., Leuven,Belgium, each mounted on a 1 ml syringe, reference BS-01N, Terumo, 15dermal papillae are extracted from the follicular bulbs.

These 15 papillae are introduced into a Petri dish 35 mm in diameter,containing 2 ml of medium 199 previously used, containing 20% foetalcalf serum Gibco, reference 10091-130. The dish is then placed in athermostated incubator at 37° C. under 5% of CO₂.

A first passage is performed after 3 weeks of incubation without havingreplaced the medium beforehand. The medium is removed, 1 ml of trypsinsolution Gibco reference 25050-022 is introduced into the Petri dish,the dish is again placed in the incubator for 4 minutes. The cells(fibroblasts of papillae) thus resuspended in trypsin solution(microscope check) are introduced into a 15 ml tube, reference Falcon,352097, Becton Dickinson, Chemin des sources, BP 37, Meylan 38241,containing 10 ml of medium 199 previously used (containing 20% foetalcalf serum).

After centrifuging for 5 minutes at 1500 revolutions per minute, thesupernatant is removed and the cell pellet is taken up in 5 ml of medium199 previously used (containing here 10% foetal calf serum).

The cell suspension is introduced into a Petri dish 35 mm in diameterand placed again in the incubator (37° C., 5% CO₂).

The following passages P2, P3, P4 are performed according to the sameprinciple. They are carried out when the cells have reached confluence

2. Extraction and Purification of the Messenger RNAs

The extraction of the messenger RNAs from the fibroblasts of dermalpapillae (performed on passages P3 or P4, 35 mm dish at confluence) iscarried out according to the protocol and with the reagents of theQuickPrepr mRNA kit (Pharmacia Biotech, Brussels, Belgium). For eachsample (a cell culture at confluence in a dish 35 mm in diameter)studied, the following protocol will be applied.

The cell culture supernatant is removed and replaced with 800 μl oflysis buffer, the lysate obtained is recovered and introduced into a 1.5ml polypropylene microtube (tube 1).

1 ml of suspension of Oligo(dT-18) cellulose microspheres is introducedinto a 15 ml microtube (tube 2) and centrifuged at 14 000 rpm for 1minute. The supernatant is removed. The content of tube 1 is thenintroduced into tube 2, the microspheres are resuspended in the lysateby gently stirring the tube for 3 minutes.

The polyA+ RNAs attached to the microspheres are isolated from thecontaminants by washing. The tube is centrifuged at 14 000 rpm for 1minute, the supernatant is removed and replaced with 1 ml of washingbuffer (high salt buffer). The microspheres are resuspended as above andthe tube is gently stirred for 1 minute. The tube is centrifuged at 14000 rpm for 1 minute, the supernatant is again removed and replaced with1 ml of low salt buffer.

In total, five washes with the low salt buffer followed by three washeswith the high salt buffer are thus carried out.

The content of the third wash (microspheres+buffer) is introduced into amicrocolumn containing a filter at the base (Microspin™ column) placedin a 1.5 ml microtube. The whole is centrifuged at 14 000 rpm for 1minute. The microcolumn is recovered and placed in a 15 ml microtube.The polyA+ messenger RNAs are eluted with a total final volume of 0.4 mlof elution buffer previously heated to 65° C.

3. Precipitation of the Messenger RNAs

10 μl of glycogen solution, 40 μl of 2.5 M potassium acetate and 1 ml ofabsolute ethanol at −20° C. are introduced into the tube containing theeluate. The tube is placed in dry ice (−80° C.). After 1 h 00 min, thetube is centrifuged at 4° C. at 17 500 rpm for 15 minutes. Thesupernatant is carefully removed (the mRNAs form a very small pellet)and replaced with 1 ml of 80% ethanol (ethanol/water; v/v) at −20° C.The tube is centrifuged for 15 min at 17 500 rpm at 4° C. and thesupernatant completely removed. The pellet is taken up in 8 μl ofsterile distilled water.

4. Synthesis of Complementary DNA (cDNA) Strands

This step is performed using the First strand cDNA synthesis kit(Pharmacia Biotech, Brussels, Belgium).

The tube containing the mRNAs is placed at 65° C. for 10 minutes andthen on ice for 5 minutes, and there are then introduced:

5 μl of a buffered solution containing a reverse transcriptasesuspension,1 μl of Oligo(dT-18) primers at 0.8 μg/ml,1 μl of aqueous dithiothreitol solution having a titre of 200 nM.

The tube is incubated at 37° C. for 1 h 00 min. The reaction is blockedby placing the tube on ice.

5. Choice of the Primers, Polymerase Chain Reaction (PCR)

1 μl (of a 1/10 dilution in sterile distilled water) of complementaryDNAs thus obtained is subjected, in a buffered medium, to a polymerasechain reaction (PCR), in the presence of specific pairs of primers(having a titre of 40 ng/ml), TAQ Polymerase and nucleotides accordingto the data from the supplier.

The primers specific for the sequences of interest which will be usedare obtained by synthesis to order by Genset SA, rue Robert et SoniaDelaunay, Paris. The first pair of primers hybridized with the sequenceencoding an ubiquitous protein (β-actin). The second pair of primershybridized with the sequence encoding 15-hydroxyprostaglandindehydrogenase.

Human β-Actin; Genbank Accession No.: NM_(—)001101

Sense primer (SEQ ID NO.:1): 5′-ATGGATGATGATATCGCCGCGCT-3′ Anti-senseprimer (SEQ ID NO.:2): 5′-CGGAGTCGTCATACTCCTGCTTG-3′Amplified fragment: 1 096 base pairs.Human 15-hydroxyprostaglandin (15-PGDH); Genbank Accession No.:NM_(—)000860

Sense primer (SEQ ID NO.:3): 5′-TGCCAATGGATTGATAACACTCAT-3′ Anti-senseprimer (SEQ ID NO.:4): 5′-ACAGCAGTTTTCATCTGGGATATG-3′Amplified fragment: 706 base pairs.

Prostaglandin F Synthase (PGFS); Genbank Accession No.: AB018580

Sense primer (SEQ ID NO.:5): 5′-AATTCCGGGCAGCAAACAT-3′ Anti-sense primer(SEQ ID NO.:6): 5′-ACACACAGGGCTTCTGGTAGACA-3′Amplified fragment: 1 061 base pairs.

The PCR reaction is carried out according to an adaptation of the TAKARATaq® protocol, TAKARA Shuzo Co. Ltd., Biomedical Group, Seta 3-4-1,Otsu, Shiga, 520-2193, Japan. The hybridization temperature is 54° C.for the pairs of primers (β-actin; PGFS, 15-PGDH), number of cycles=35.

The following are introduced into a microtube suitable for PCR:

1 μl (a 1/10 dilution) of complementary DNA,

43 μl of the mixture of nucleotides in buffered solution*,

5 μl (2.5 μl+2.5 μl) of pairs of primers at 40 ng/μl are in reaction,

50 μl of mineral oil. * A buffered nucleotide solution is prepared bymixing 171 μl of sterile distilled water, 24.5 mil of 10× buffer fromthe kit and 20 μl of nucleotide (dNTP) mixture from the kit.

The tube is placed in a PCR apparatus and the following cycles areprogrammed:

 4′ at 95° C.  1 cycle 30″ at 94° C.  1′ at 54° C. 35 cycles  1′ at 72°C.  7′ at 72° C.  1 cycle

6. Reading to 6.1 Preparation of an Agarose Gel

Weighing of 0.65 g of agar (Molecular biology certified agarose, Bio-Radlaboratories 2000 Alfred Nobel Dr, Hercules, Calif. 94547, USA).

Addition of 50 ml of 1×TAE buffer, Amresco, Solon, Ohio 44139, USA.

The agarose in suspension is heated to boiling temperature and thenintroduced into a tank containing a drop of ethydium bromide (25 μg),Amresco, Solon, Ohio 44139, USA.

A “comb” allowing the deposition of samples is placed at one end of thetank. After cooling for 30 minutes (room temperature), 20 μl of the PCRresults are individually introduced into a well of the gel, as well as10 μl of a mixture of molecular weight standards (Amplisize, molecularRuler, 170-8200, Bio-Rad laboratories 2000 Alfred Nobel Dr., Hercules,Calif. 94547, USA).

The whole is subjected, immersed in a large excess of 1×TAE buffer, toan electrical field of 100 volts for 45 minutes.

Exposure of the gel under ultraviolet light makes it possible to observethe results obtained by fluorescence.

Weight of the amplimers expectedActin=1 096 base pairs, 15-PGDH=707 base pairs,PGFS=1 061 base pairs

Samples 1, 2, 3 are obtained from different cultures of fibroblasts ofdermal papillae of human hair.

Test for the Expression of 15-Hydroxyprostaglandin Dehydrogenase

The results are presented in FIG. 1.

It is observed that 15-PGDH is expressed in the different samples, witha characteristic MW band at 706 bp

Test for the Expression of Prostaglandin F Synthase

The results are presented in FIG. 2.

It is observed that PGFS is expressed in the different samples, with acharacteristic 1 061 pb MW band.

EXAMPLE 2 Cloning from Fibroblasts of Dermal Papillae of Hair and thenPurification of PGFS and 15-PGDH

The extraction and the purification of the poly-A+ messenger RNAs andthen the synthesis of complementary DNA (cDNA) were carried out using aculture of fibroblasts of dermal papillae of hair as described inExample 1

Pairs of primers (synthesized by Genset) to which were added sequencesencoding restriction sites were chosen for 15-PGDH (Genbank accessionNo. NM_(—)000860) and for PGFS (Genbank accession No.=AB018580).

a) Primers for 15-PGDH (SEQ ID NOS.: 7&8)

5′-GGG GAT CCA TGC ACG TGA ACG GCA AAG TG-3′,sense primer BamH1 site (in bold)

5′-TCT CGA GAG CTG TTC ATT GGG T-3′;anti-sense primer Xho1 site (in bold)

b) Primers for PGFS (SEQ ID NOS.: 9&10)

5′-CGG GAT CCA TGG ATT CCA AAC AGC ACT GTG-3′;sense primer BamH1 site (in bold)

5′-CG GAA TTC TTA ATA TTC ATC TGA A-3′;anti-sense primer EcoR1 site (in bold)

c) Polymerase chain reaction (PCR)

The PCR protocol applied for the cloning of 15-PGDH and for the cloningof PGFS is broadly similar to that described above, except for thedifferences below:

The Taq Polymerase used according to the manufacturer's data (Pfu Turbo®DNA Polymerase), Stratagene cloning systems, 11011 North Torrey PinesRoad, La Jolla, Calif. 92037

Hybridization temperature 59° C., extension time 2 min, 25 cycles intotal for 15-PGDH, expected amplimer=815 bp.

Hybridization temperature 50° C., extension time 2 min, 25 cycles intotal for PGFS, expected amplimer 989 bp.

d) The PCR products are digested with the restriction enzymes (BamH1;Xho1 for 15-PGDH and BamH1; EcoR1 for PGFS) according to themanufacturer's data (Amersham Pharmacia Biotech, 12 avenue desTropiques, ZA Courtaboeuf, 91944 Les Ulis) and then individually run ona 1.3% agarose gel (see Example 1, 6. Reading).

e) Cutting out the bands corresponding to the expected amplimers (seeac) using a scalpel (after locating under ultraviolet light) andpurification of these cutouts according to the recommendations of themanufacturer of the Wizard® PCR Preps DNA Purification system kit(Promega Corporation, 2800 Woods Hollow Road, Madison, Wis. 53711-5399).

f)

15-PGDH

Ligation into a vector pGEX 413 (Amersham Pharmacia Biotech, 12 avenuedes Tropiques, ZA Courtaboeuft 91944 Les Ulis) previously digested(Bam1/Xho1) and purified, according to the information from themanufacturer of the Fast-Link™ DNA ligation kit, Epicentre, 1202 AnnStreet, Wis. 53713.

PGFS

Ligation into a vector pGEX 2T (Amersham Pharmacia Biotech, 12 avenuedes Tropiques, ZA Courtaboeuf, 91944 Les Ulis) previously digested(BamH1/EcoR1) and purified, according to the information from themanufacturer of the Fast-Link™ DNA Ligation kit, Epicentre, 1202 AnnStreet, Wis. 53713. These two vectors allow the synthesis of the proteinof interest coupled to a fusion protein (glutathione sulphotransferase).The fusion protein will allow subsequent purification of the protein ofinterest.

g) Transformations

Competent bacteria of the BL21DE3plys type will be used for thetransformation with the construct (pGEX4T3/15-PGDH), competent bacteriaof the BL21DE3 type for the transformation with the construct (pGEX2T/PGFS). These two strains are marketed by the company Stratagene. Thetransformations are carried out according to a protocol which isconventionally applied as described for example in the Fast-Link™ DNAligation kit previously used. The infected bacteria (clones) areselected (white colonies) after deposition and culture for 24 h 00 minat 37° C. of a fraction of these transformation products on LB-Agarmedium poured into a Petri dish (L-2897, Sigma-Aldrich, L'isle D'AbeauChesne, BP 701, 38297, Saint Quentin Fallavier) containing 100 μg/ml ofampicillin.

h) Production of 15-PGDH and PGFS.

A colony obtained from the “15-PGDH transformation” Petri dish iscollected and introduced into 250 ml of LB medium (1-3022,Sigma-Aldrich, L'isle D'Abeau Chesne, BP 701, 38297, Saint QuentinFallavier) containing 100 μg/ml of ampicillin, the flask being incubatedwith stirring for 16 h 00 min at 37° C.

The same procedure is carried out in another flask, for a colonyobtained from the “PGFS transformation” Petri dish.

After 16 h 00 min, each of the flasks is introduced into an Erlenmeyerflask containing 2.5 l of LB medium containing 100 μg/ml of ampicillin.These two Erlenmeyer flasks are incubated, with stirring, at 37° C. for3 h 00 min to 4 h 00 min until the optical density, measured at 630 nm,is between 0.6-0.9).

Addition of isopropyl β-D-thiogalactopyranoside (IPTG), (16758,Sigma-Aldrich, L/isle D'Abeau Chesne, BP 701, 38297, Saint QuentinFallavier) such that the final concentration is 0.1 mM.

The Erlenmeyer flasks are incubated for an additional 24 h 00 min, withstirring, at room temperature (20-25° C.). The cultures thus obtainedare centrifuged (in 250 ml fractions) at 5 000 rpm for 7 minutes, thepellets are taken up in 3 ml of 10 mM phosphate buffer pH=7.00 (4° C.)containing a mixture of protease inhibitors (protease inhibitor cocktailset 1539131, Calbiochem-Novabiochem Corporation, 10394 Pacific CenterCourt, San Diego, Calif. 92121). The bacterial suspensions (groupedtogether into fractions of 40 ml in a polypropylene tube) are blocked onice and subjected to ultrasound (Vibra cell 20 kHz, 72434, 20 Bioblockscientific, Parc d'innovation, BP111, 67403 Illkirch) the probe beingdipped into each tube for 15 seconds (6 shocks of 15 seconds per tube).Each tube is centrifuged at 4° C., 16 000 rpm, for 1 h 00 min.

i) Purification of 15-PGDH and PGFS

The supernatants are recovered and introduced into a polypropylene tubecontaining 1 ml of Glutathione-Sepharose® 4B (40 ml of supernatant per 1ml of Glutathione-Sepharose® 4B) previously washed according to the torecommendations of the manufacturer (Amersham Pharmacia Biotech, 12avenue des Tropiques, ZA Courtaboeuf, 91944 Les Ulis).

The tubes are placed vertically on a rotary shaker, rotation 10revolutions per minute for 1 h 00 min at room temperature (20-25° C.).

The tubes are centrifuged at 1 000 rpm for 3 minutes, and thesupernatant is removed.

40 ml of a 10 mM phosphate buffer pH=7.00 are introduced into each ofthe tubes. After gentle stirring (inverting) the tubes are againcentrifuged for 3 minutes at 1 000 rpm.

The operation is performed 5 times. A sixth wash is performed with 40 mlof phosphate buffered saline pH=7.2 (PBS, Bio-Merieux S, 69280Marcy-l'Etoile). After centrifugation, the supernatant is again removed.

j) Elution of 15-PGDH and PGFS

A suspension of thrombin protease is reconstituted at 1 unit/μl in PBSaccording to the recommendations of the manufacturer (Amersham PharmaciaBiotech, 12 avenue des Tropiques, ZA Courtaboeuf, 91944 Les Ulis).

950 μl of PBS and 50 μl of reconstituted thrombin suspension areintroduced into each tube containing 1 ml of Glutathione-Sepharose® 4B.They are stirred in a slightly inclined position, 16 h 00 min at 250rpm. After 16 h 00 nm in, the tubes are centrifuged at 3 000 rpm for 5min and the supernatants are recovered. The quantity of protein isevaluated according to the Bio-Rad DC Protein Assay procedure (Bio-RadLaboratories, 2000 Alfred Nobel Dr, Hercules, Calif. 94547).

Thus, for 15-PGDH as well as for PGFS, between 0.2 and 5 mg of proteinare obtained per ml, most often 1 mg/ml.

The protein suspensions thus obtained are diluted respectively in PBSsupplemented with 10% glycerol and PBS such that the final proteinconcentrations are 0.2 mg/ml for 15-PGDH and 0.5 mg/ml for PGFS. Thesuspensions are blocked at −80° C. until used.

Electrophoretic analyses (SDS-Page) performed under standard conditionsdemonstrate the quality of the results thus obtained. These results arepresented in FIG. 3.

EXAMPLE 3 Evaluation of the Effect of Molecules on these Enzymes andCharacterization of Certain Families of Molecules as Specific 15-PGDHInhibitors

a) 15-PGDH Test

The enzyme obtained is at the concentration of 0.3 mg/ml and is blockedat −80° C. This suspension is thawed and stored on ice.

Preparation of a 100 mM Tris buffer pH=7.4 containing 0.1 mMdithiothreitol (D5545, Sigma-Aldrich, L'isle D'Abeau Chesne, BP 701,38297, Saint Quentin Fallavier), 1.5 mM β-NAD (N6522, Sigma-Aldrich,L'isle D'Abeau Chesne, BP 701, 38297, Saint Quentin Fallavier), 50 μMProstaglandin E2 (P4172, Sigma-Aldrich, L'isle D'Abeau Chesne, BP 701,38297, Saint Quentin Fallavier).

0.965 ml of this buffer (previously heated to 37° C.) is introduced intoa spectrophotometer cuvette (Perkin-Elmer, Lambda 2) thermostated at 37°C., whose wavelength for measurement is set at 340 nm. 0.035 ml ofenzymatic suspension at 37° C. are introduced into the cuvetteconcomitantly with the recording (increase in the optical density at 340nm).

The maximum reaction rate is recorded.

The test values (molecules) are compared with the control value (with nomolecule), the results are expressed as % of the control value.

b) PGFS Test

The enzyme obtained is at a concentration of 0.5 mg/ml and blocked at−80° C. This suspension is thawed and stored on ice.

Preparation in a brown flask (protection from light) of a 100 mM Trisbuffer pH=6.5 containing 20 μM 9,10-phenanthrenequinone** (P2896,Sigma-Aldrich, L'isle D'Abeau Chesne, BP 701, 38297, Saint QuentinFallavier) and 100 μM β-NADPH (N1630, Sigma-Aldrich, L'isle D'AbeauChesne, BP 701, 38297, Saint Quentin Fallavier). ** A stock solutionhaving a titre of 1 mM is prepared in absolute ethanol heated to 40° C.,the flask is placed in an ultrasonic tank in order to facilitate thesolubilization of the product.

0.950 ml of this buffer (previously heated to 37° C.) is introduced intothe cuvette of a spectrophotometer (Perkin-Elmer, Lambda 2) thermostatedat 37° C. whose wavelength for measurement is set at 340 nm, 0.05 ml ofenzymatic suspension at 37° C. is introduced into the cuvetteconcomitantly with the recording (reduction in the optical density at340 nm).

The maximum reaction rate is recorded

The test values (molecules) are compared with the control value (with nomolecule), the results are expressed as % of the control value.

The results obtained for the molecules A and B are the following.

% Inhibition % Inhibition at 50 μM 15-PGDH PGFS Molecule A:5-amino-4,6-di- 43  5 chloro-2-phenylpyrimidine Molecule B:N-[7-(2-chloro- 57 10 phenyl)-5-oxo-5,6,7,8-tetra-hydroquinazolin-2-yl]benzamide

EXAMPLE 4 Demonstration of the Inhibitory Activity of the Compounds ofFormula (I) or (II)

The test values (containing the compounds (I) or (II)) are compared withthe control value (with no compound (I) or (II)) according to theprotocol described in Example 3; the results indicated represent the %inhibition for a given concentration of the compound (I) or (II).

Com- Inhibition of 15- pound Structure PGDH at 50 μM 1

70% inhibition 4

60% inhibition

It is evident from this table that the compounds 1 and 4 are indeed15-PGDH inhibitors. They will therefore allow a reduction in hair loss,in particular which is natural.

EXAMPLE 5 Characterization of Inhibitors Specific for 15-PGDH

The following compounds are tested according to the protocol of Example3.

-   N-(2-Methoxyphenyl)-2-[5-phenyl-2H-tetrazol-2-yl]acetamide (compound    4)-   1-Phenyl-2-(2-phenyl-2H-tetrazol-5-yl)ethanone (compound 1)-   N-(4-Methylphenyl)-2-{5-[3-trifluoromethyl)phenyl]-2H-tetrazol-2-yl}acetamide    (compound R29A)

The concentrations of the test compounds causing a 50% inhibitionrespectively for the two enzymes are presented below

15-PGDH PGFS Compound Tested (IC50 μM) (IC50 μM)

55 >75

30 >75

40 >75

The test compounds are inhibitors specific for 15-PGDH. In this regard,they will therefore allow a reduction in hair loss, in particular whichis natural.

EXAMPLE 6 Demonstration of the Efficacy of a Specific 15-PGDH Inhibitoron a Cellular Model

The present study aims to evaluate this selection of compounds in acellular model. This will provide us with information on the penetrationof the active agent into the cytosol and on its efficacy as an inhibitorwhich is selective for 15-PGDH under more complex experimentalconditions than a simple reaction medium.

Materials and Methods

D-2. Culture of U937 (CRL-1593 American Type Cells Collection) in RPMI1640 medium+10% foetal calf serum+2 mM L-Glutamine+antibiotics at 37° C.under 5% CO₂.

D-1. Preparation of a suspension of U937 (1×10⁶ cells/ml) in RPMI 1640medium+10% foetal calf serum+2 mM Glutamine+Antibiotics+10 nM PMA(phorbol 12-myristate 13-acetate), introduction of 200 μl/well of thissuspension into a 96-well plate (3 wells per molecule and perconcentration to be tested+corresponding controls). Incubation 36 h 00min at 37° C. under 5% CO₂.

J0. Removal of the supernatants (the cells have adhered: microscopecheck), and introduction into each well of 100 μl of RPMI 1640+2 mML-Glutamine+10 ng LPS (except absolute control)+the molecule to betested at the desired concentration (here 5 and 25 μM).

Incubation for 6 h 00 min at 37° C. under 5% CO₂.

The stock solutions of molecules to be tested are at 25 mM in DMSO.

All the wells will contain the same final quantity of DMSO.

Immediate evaluation of the quantity of PGF2α secreted by the cells (50μl) under different conditions (molecules or controls) using animmunoenzymatic assay kit (Cayman ref. 516011).

Results as % of the LPS Control

Reference Molecule (5 μM) % of the control Compound 1:1-phenyl-2-(2-phenyl- +16 2H-tetrazol-5-yl)ethanoneN-(4-methylphenyl)-2-{5-[3- +46 (trifluoromethyl)phenyl]-2H-tetrazol-2-yl}acetamide Compound 4: N-(2-methoxyphenyl)-  +62-(5-phenyl-2H-tetrazol-2- yl)acetamide

This confirms that the inhibitors selective for 15-PGDH are effective ina cellular environment and protect prostaglandins.

The compositions below are obtained by the usual techniques commonlyused in the cosmetic or pharmaceutical field.

EXAMPLE 7 Hair Lotion

Compound 1 0.80 g Propylene glycol 10.00 g Isopropyl alcohol qs 100.00 g

This lotion is applied to the scalp, once or twice per day, at the rateof 1 ml per application, by lightly massaging the scalp in order tocause penetration of the active agent. The hair is then dried in theopen air. This lotion makes it possible to reduce the loss of hair andto promote its regrowth.

EXAMPLE 8 Hair Lotion

Compound 4 1.00 g Propylene glycol 30.00 g Ethyl alcohol 40.00 g Waterqs 100.00 g

This lotion is applied to the scalp, once or twice per day, at the rateof 1 ml per application by lightly massaging the scalp in order to causepenetration of the active agent. The hair is then dried in the open air.

1. A cosmetic or pharmaceutical composition comprising at least one15-PGDH inhibitor which is a tetrazole compound of formula (I) or (II):

an inorganic or organic salt thereof, wherein each of R₁ and R₂ ishydrogen or halogen; R₃ is NR₆R′₆, or R₃ is naphthyl or phenyloptionally substituted with OR₉, R₄ is naphthyl or phenyl, optionallysubstituted with OCH₃ or CF₃; R₆ is hydrogen; R′₆ is phenyl optionallysubstituted with OR₉; and R₉ is saturated, linear or branched C₁-C₁₀alkyl; and physiologically acceptable excipients therefor.
 2. A cosmeticor pharmaceutical composition according to claim 1, wherein the compoundof formula (I) or (II) is:1-phenyl-2-(2-phenyl-2H-tetrazol-5-yl)ethanone having the formula:

1-(2-methoxyphenyl)-2-(2-phenyl-2H-tetrazol-5-yl)ethanone having theformula:

N-(4-methylphenyl)-2-{5-[3(trifluoromethyl)phenyl]-2H-tetrazol-2-yl}acetamide having the formula:

N-(2-phenyl)-2-(5-phenyl-2H-tetrazol-2-yl)acetamide having the formula:

(2-methoxyphenyl)-2-(5-phenyl-2H-tetrazol-2-yl)acetamide having theformula:


3. A cosmetic or pharmaceutical composition comprising at least one15-PGDH inhibitor selected from the group consisting of: Molecule A:5-Amino-4,6-dichloro-2-phenylpyrimidine having the formula

Molecule B:N-[7-(2-Chlorophenyl)-5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl]benzamidehaving the formula:

1-Phenyl-2-(2-phenyl-2H-tetrazol-5-yl)ethanone having the formula:

N-(4-Methylphenyl)-2-{5-[3-(trifluoromethyl)phenyl]-2H-tetrazol-2-yl}acetamidehaving the formula:

N-(2-Methoxyphenyl)-2-(5-phenyl-2H-tetrazol-2-yl)acetamide having theformula:

and physiologically acceptable excipients therefor.
 4. A compositionaccording to claim 1, wherein said at least one 15-hydroxyprostaglandindehydrogenase inhibitor is encapsulated in a structure selected from thegroup consisting of microspheres, nanospheres, oleosomes andnanocapsules.
 5. A composition according to claim 2, wherein said atleast one 15-hydroxyprostaglandin dehydrogenase inhibitor isencapsulated in a structure selected from the group consisting ofmicrospheres, nanospheres, oleosomes and nanocapsules.
 6. A compositionaccording to claim 3, wherein said at least one 15-hydroxyprostaglandindehydrogenase inhibitor is encapsulated in a structure selected from thegroup consisting of microspheres, nanospheres, oleosomes andnanocapsules.
 7. A cosmetic or pharmaceutical composition according toclaim 1, wherein the 15-PGDH inhibitor is an inhibitor specific for15-PGDH and wherein the ratio between the 15-PGDH inhibiting activityand the prostaglandin F synthase (PGF synthase) inhibiting activity isgreater than
 1. 8. A cosmetic composition according to claim 1, in theform of a hair cream, a hair lotion, a shampoo, an after-shampoo, a hairmascara or an eyelash mascara.
 9. A cosmetic composition according toclaim 2, in the form of a hair cream, a hair lotion, a shampoo, anafter-shampoo, a hair mascara or an eyelash mascara.
 10. A cosmeticcomposition according to claim 3, in the form of a hair cream, a hairlotion, a shampoo, an after-shampoo, a hair mascara or an eyelashmascara.